First Reported Nonpeptide AT1 Receptor Agonist (L-162,313) Acts as an AT2 Receptor Agonist in Vivo

Article abstract of DOI:10.1021/jm031031i

In this investigation, it is demonstrated that the first nonpeptide AT ₁ receptor agonist L-162,313 (1), disclosed in 1994, also acts as an agonist at the AT₂ receptor. In anesthetized rats, administration of compound 1 intravenously

Full text of DOI:10.1021/jm031031i

1536
J . Med. Chem. 2004, 47, 1536-1546
F ir st Rep or ted Non p ep tid e AT1 Recep tor Agon ist (L-162,313) Acts a s a n AT2
Recep tor Agon ist in Vivo
Yiqian Wan,† Charlotta Wallinder,† Berndt J ohansson,‡ Mathias Holm,‡ Mahalingam A. K.,† Xiongyu Wu,†
Milad Botros,‡ Anders Karle´n,† Anders Pettersson,‡ Fred Nyberg,§ Lars Fa¨ndriks,‡ Anders Hallberg,† and
Mathias Alterman*,†
Department of Medicinal Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden, Department of
Gastrosurgical Research, Sahlgrenska Academy at Gothenburg University, SE-413 45 Gothenburg, Sweden, and Department of
Biological Research on Drug Dependence, BMC, Uppsala University, P.O. Box 591, SE-751 23 Uppsala, Sweden
Received September 12, 2003
In this investigation, it is demonstrated that the first nonpeptide AT1 receptor agonist
L-162,313 (1), disclosed in 1994, also acts as an agonist at the AT2 receptor. In anesthetized
rats, administration of compound 1 intravenously or locally in the duodenum increased duodenal
mucosal alkaline secretion, effects that were sensitive to the selective AT2 receptor antagonist
PD-123,319. The data strongly suggest that 1 is an AT2 receptor agonist in vivo. To the best of
our knowledge, this substance is the first nonpeptidic low-molecular weight compound with
an agonistic effect mediated through the AT2 receptor.
In tr od u ction
Selective AT1 receptor antagonists block well-known
functional effects of the octapeptide angiotensin II (Ang
II), such as vasoconstriction, aldosterone release, and
cardiovascular growth.1 In 1994, the first nonpeptide
Ang II agonist, L-162,313 (1) was disclosed (Figure 1).2
At that time, nonpeptide agonists of peptide receptors
were rare, being confined largely to agonists of opioid
receptors. Notably, removal of a methyl group from
L-162,782, an agonist closely structurally related to the
thiophene derivative 1, provided L-162,389 which was
found to act as an antagonist in vivo.3 Thus, a subtle
molecular alteration was shown to determine the ago-
nist/ antagonist properties of these ligands. The three
compounds described above were all essentially equi-
potent as AT1/AT2 receptor binding ligands, but by
replacing the alkyl group with a m-methoxybenzyl
F igu r e 1.
group, an agonist, L-163,491, with an approximately 70-
fold selectivity for the AT1 receptor subtype, was
obtained.4
part of 1 (Series A, Figure 2) and (b) to determine the
receptor selectivities after retaining the lower part of 1
but replacing the bicyclic imidazopyridine ring system
with substituted quinazolinones, as the latter structure
is found in a large number of selective AT2 receptor
antagonists (Series B, Figure 2).10
We herein report that the AT1 receptor agonist 1, the
compound with the most favorable AT2/AT1 affinity ratio
in the A series acts as an AT2 receptor agonist in the
animal model. In contrast, compound 12 in series B,
with a 40-fold greater affinity for the AT2 receptor
versus the AT1 receptor, exerts no agonistic effects in
this in vivo model.
Ang II exhibits a similar binding affinity to the AT1
and AT2 receptors, sharing a sequence homology of only
32-34% at the amino acid level in rat.5 We felt encour-
aged to answer the question of whether 1 also would
exert agonistic properties toward the AT2 receptor.5,6
Recently, we reported an AT2 receptor-mediated Ang
II stimulation of duodenal mucosal alkaline secretion
in the rat,7 an effect that could be blocked by the AT2
selective antagonist PD-123,3198 (35) but not by the AT1
receptor antagonist losartan.9 Prior to selecting the
compounds for studies in this animal model, we decided
to (a) assess the impact on AT1/AT2 receptor affinity
caused by minor alterations of the sulfonylcarbamate
Ch em istr y
The thiopheneboronic acid 2, a key intermediate for
the synthesis of the compounds in both series, was
prepared essentially as described by Kevin et al.11 Thus,
thiophene-2-sulfonyl chloride was first converted to the
N-tert-butylsulfonamide. Subsequent alkylation followed
* Corresponding author. Tel: +46-18-4714905. Fax: +46-18-
4714474. E-mail: mathias.alterman@orgfarm.uu.se.
† Department of Medicinal Chemistry, Uppsala University.
‡ Sahlgrenska Academy at Gothenburg University.
§ Department of Biological Research on Drug Dependence, Uppsala
University.
10.1021/jm031031i CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/14/2004

AT2 Receptor Agonist in Vivo
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1537
F igu r e 2.
Sch em e 1a
a
Reagents and conditions: (a) Pd(PPh3)4, NaOH (aq), ethanol/toluene; (b) TFA; (c) alkyl chloroformate or acyl chloride, pyrrolidinopy-
ridine, pyridine.
Ta ble 1. Series A
by selective 3-lithiation/boration delivered 2. A Suzuki
coupling of 3-(4-bromobenzyl)-2-ethyl-5,7-dimethyl-3H-
imidazol[4,5-b]pyridine12 with the boronic acid 211 pro-
vided 3 in good yield. Deprotection by TFA, to give the
primary sulfonamide followed by reactions with alkyl
chloroformates and acyl chlorides, afforded 12, 4-6, and
7-9, respectively (Scheme 1).
A Suzuki coupling of 2 with 3-(4-bromobenzyl)-6-nitro-
2-propyl-3H-quinazolin-4-one yielded 10 in high yield.
The ethyl group most often found in the 2-position of
the quinazolin-4-one scaffold of AT2 receptor antagonists
was replaced with propyl since Glinka et al. had
observed that the propyl group in a related series of
compounds gave higher AT2 receptor affinities when
combined with the sulfonamide based carboxylic acid
bioisostere.10c
The compounds in series B were prepared as outlined
in Scheme 2. Deprotection of 10 and reaction with butyl
chloroformate gave 11 in a good yield. To obtain the
derivatives 12-21, the nitro group of 10 was first
reduced using ammonium formate and palladium on
charcoal to provide 22. Acylation of the amine function
of 22 with acetyl chloride, benzoyl chloride, and thienoyl
chloride respectively afforded compounds 23-25 which
were debutylated and then reacted with butyl chloro-
formate to yield the secondary amides 12-14. The
tertiary amides 15-21 were prepared by reductive
alkylation using acetaldehyde or benzaldehyde with
triacetoxyborohydride as reducing agent to give 26 and
27, respectively. These secondary amines were there-
after acylated to afford 28-34 and subsequently depro-
tected and treated with butyl chloroformate to deliver
the desired tertiary amides.
Bin d in g Assa ys. Compounds 1, 4-9, 11-21 were
evaluated in radioligand-binding assays by displace-
ment of [125I]Ang II from AT1 receptors in rat liver
membranes and from AT2 receptors in pig uterus
membranes in essence as described previously (Table
1).13 The natural substrate Ang II, the selective AT1
a
Ki values are an average from three determinations. Standard
deviations are less than 15% in all cases.
receptor antagonist losartan,9 and the selective AT2
receptor antagonist 358 were used as reference sub-
stances.
In Vivo Assa ys. The in vivo experiments were
performed on anaesthetized nonfasted male Sprague-
Dawley rats. A femoral artery and one or two veins were
catheterized for subsequent blood pressure measure-
ments and drug infusions, respectively. Duodenal mu-

1538 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wan et al.
Sch em e 2a
a
Reagents and conditions: (a) Pd(PPh3)4, NaOH (aq), ethanol/toluene; (b) TFA; (c) butyl chloroformate, pyrrolidinopyridine, pyridine;
(d) Pd/C, HCO2NH4, MeOH; (e) acyl chloride, DIEA, CH2Cl2; (f) acetaldehyde or benzaldehyde, NaB(OAc)3H, AcOH, CH2ClCH2Cl.
-
cosal alkaline secretion (HCO3 secretion) was mea-
sured by a pH-stat titration technique.14 Alkaline
secretion to the perfusate was continuously titrated to
pH 7.4 with 0.02 M HCl controlled by a pH-stat device.
5-position, as reported by Kevin et al.,11a in that even
the small structural modifications performed tended to
reduce AT2 receptor affinity while retaining AT1 recep-
tor affinity.
The most potent AT2 receptor ligand in series A, i.e.
1 (not receptor subtype selective but a proven partial
AT1 receptor agonist) was investigated in the rat in vivo.
Duodenal mucosal alkaline secretion in rats has been
shown previously to be inhibited by AT1 receptor activa-
tion and increased after AT2 receptor stimulation.7,16
As shown in Figure 3, intravenous administration of
1 (bolus 0.3 mg/kg plus 30 µg/kg×h) increases mean
arterial pressure by approximately 10 mmHg. This
pressor effect was reversed, with a markedly lowered
arterial pressure by addition of the AT1-receptor an-
tagonist losartan (10 mg kg-1 iv bolus) indicating that
1 activates AT1 receptor-mediated vasoconstriction.
Interestingly, the compound alone also increased the
duodenal mucosal alkaline secretion moderately. The
addition of losartan markedly increased this secretory
stimulation (Figure 3). When losartan was combined
with the AT2 receptor antagonist 35 this effect was
absent (data not shown, n ) 3). Although a proper dose-
response-curve was not performed with regard to in-
travenous administration, the effect on mucosal alkaline
secretion by compound 1 at the presently used infusion
rate can be considered of the same order of magnitude
as previously reported for the peptide compounds Ang
Resu lts a n d Discu ssion
The results obtained in the AT1 and AT2 receptor
binding assays are presented in Tables 1 and 2. The Ki
values for compound 1 were determined as 3.9 nM for
the AT1 and 2.8 nM for the AT2 receptors (lit. data
IC50: 1.1 nM and 2.0 nM, respectively2b). As apparent
from Table 1 all variations of the butyloxy group
investigated were deleterious with respect to AT2 recep-
tor affinity, although low affinity was encountered by
shortening of the side-chain by removal of the oxygen
atom (compound 9). Notably, the ethyloxy derivative 4
exhibited no detectable AT2 receptor affinity. The lack
of AT2 receptor affinity exhibited by 7 is also somewhat
remarkable considering the previous results by Mantlo
et al.,15 who in their series of compounds observed a
higher selectivity for the AT2 receptor with compounds
bearing the cyclopentyl side-chain. With regard to AT1
receptor affinity, the impact of alterations to the buty-
loxy side-chain was considerably less pronounced. All
compounds bound in varying degree to the AT1 receptor.
Thus, the effect caused by alterations of the butyloxy-
carbonylsulfonamide portion of 1 seemed to resemble
the outcome of variations of the isobutyl group in the

AT2 Receptor Agonist in Vivo
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1539
Ta ble 2. Series B
F igu r e 3. Anesthetized Sprague-Dawley rats (n ) 5). Effects
of intravenous administration of 1 (bolus 0.3 mg/kg plus 30
µg/kg × h) and later addition of the AT1 receptor antagonist
losartan (10 mg/kg bolus iv) on duodenal mucosal alkaline
secretion and mean arterial pressure.
F igu r e 4. Anesthetized Sprague-Dawley rats. Effects of local
(intra duodenal) administration of 1 at consecutively increased
concentrations in 30 min periods in absence (n ) 5) and in
the presence (n ) 5) of 35 (0,1 mM). Values represent means
of the last 15 periods of each concentration.
conceivably provide high AT2 receptor selectivity. We
were particularly attracted by a series of AT2 receptor
ligands of the biaryl type reported by Glinka et al.10
Some of these compounds exhibited an impressive AT2/
AT1 selectivity (e.g. for one of the derivatives, AT1: Ki
>3000 nM and AT2: Ki ) 0.06 nM). The compounds
were comprised of a quinazolin-4-one scaffold substi-
tuted in the 6-position with, for example, a variety of
acylamido groups. Partly guided by the results of Glinka
et al., the compounds in Table 2 were designed and
synthesized.
From Table 2 it is apparent that the substituent in
the 6-position of the quinazolinones can significantly
affect the biological activity. While all amides (12-21)
bind to the AT1 receptor, albeit weakly, minor structural
variations could have a dramatic influence on the
affinity to the AT2 receptor. The thiophene and the
corresponding benzene rings in 1 and L-162,782, re-
spectively, act as true bioisosteres, but these aromatic
nuclei are not necessarily interchangeable when con-
stituting a part of the 6-substituent of the quinazolinone
moiety. A comparison of the tertiary ethyl amides 16
and 17 suggests that interchanging the two aromatic
systems has no large impact on the binding affinity to
the AT2 receptor. However, substitutions of the ethyl
group of the thiophene derivative 17 with either hydro-
gen (cf. 17 and 14) or a benzyl group (cf. 17 and 21) are
deleterious for AT2 receptor affinity. However, when the
benzene analogue 16 was subjected to the same modi-
II and CGP112A.7 The results support the view that
compound 1 is an unselective agonist at both the AT1
and AT2 receptors.
However, to investigate the AT2 agonistic properties
of 1 in greater depth, topical administration was em-
ployed by administering the compound in the duodenal
intraluminal perfusate. The rationale behind this mode
of administration was that AT2 receptors, but not AT1
receptors, are localized to the secreting epithelium7
making simultaneous administration of an AT1 receptor
antagonist unnecessary. It was observed that 1 given
in the perfusate raised the alkaline secretion in a
concentration dependent manner (Figure 4). Simulta-
neous presence of 35 (0.1 mM) significantly inhibited
this effect indicating that an AT2 receptor-mediated
effect was occurring. Such local intraluminal adminis-
tration of drugs did not influence arterial pressure (data
not shown). Taken together these in vivo tests strongly
suggest that 1 is a dual AT1/AT2 receptor agonist.
Since the data in series A and previous findings3
suggested that the 2-(N-butyloxycarbonyl)sulfonamide
and 5-isobutyl were compulsory for fair AT2 receptor
affinity to be achieved, we turned our attention to
modifications of the bicyclic upper part of the molecule
in the hope of improving the AT2/AT1 ratio. We hypoth-
esized that the lower part of 1 might be pivotal for
agonism while a proper upper heterocyclic moiety could

1540 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wan et al.
chromatography was performed on silica gel 60 (0.04-0.063
mm, E. Merck). Thin-layer chromatography was performed on
precoated silica gel F-254 plates (0.25 mm, E. Merck) and was
visualized with UV light. Analytical RP-LC/MS was performed
on a Gilson HPLC system with a Zorbax SB-C8, 5 µm 4.6 ×
50 mm (Agilent Technologies) column, with a Finnigan AQA
quadropole mass spectrometer at a flow rate of 1.5 mL/min
(H2O/CH3CN/0.05% HCOOH). All the organic phases were
dried over MgSO4. All chemicals were purchased from com-
mercial suppliers and used directly without further purifica-
tion.
fications, surprisingly, the affinity was largely retained
(cf. 16, 13, and 20). These results are difficult to
rationalize but demonstrate a complex structure-activ-
ity relationship of the compounds in series B. Thus, as
in series A where the AT2 affinity drops significantly
after minor structural changes to the oxybutyl chain of
the prototype compound 1 or of the isobutyl group, as
previously shown,11a a similar response is encountered
after the “bioisosteric” phenyl to thienyl exchange,
conducted in the series B.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Com -
p ou n d s 4-9. Compound 3 (50 mg, 0.10 mmol) and anisole
(150 µL) were dissolved in TFA (5 mL) and stirred overnight.
The reaction mixture was evaporated under vacuum and
coevaporated with acetonitrile twice. The residue was dissolved
in pyridine (1 mL), and pyrrolidinopyridine (15 mg, 0.10 mmol)
was added. The solution was cooled on an ice bath, and the
acyl chloride (20 equiv) was added under a N2 (g) atmosphere.
The reaction was stirred at ambient temperature overnight.
The solvent was removed under vacuum and coevaporated
with acetonitrile twice. The residue was dissolved in ethyl
acetate (50 mL) and washed with citric acid (10% aq) and
water. The organic phase was dried, evaporated, and purified
by column chromatography to give the pure compounds 4-9.
One of the most potent and AT2 receptor selective
ligands in series B, the secondary amide 12 (the ligand
with the most favorable solubility properties in the
series), was selected for in vivo studies in rat. Compound
12 was not soluble in ethanol unlike 1. Ethanol exerts
only marginal effects on the studied secretory variable
at the used concentration (<4%). DMSO was the only
solvent that could dissolve compound 12 in sufficient
concentration for evaluation. Unfortunately, DMSO was
found to exert stimulatory effects on mucosal alkaline
secretion in concentrations below 5%. However, no
additional stimulatory effect of the 40-fold AT2 receptor
selective quinazolinone compound 12 at concentrations
1-100 µM was observed in the rat duodenum, suggest-
ing that 12, contrary to 1, does not act as an AT2
receptor agonist under these test conditions. Although,
we cannot rule out that the lack of agonistic effect of
compound 12 in the rat is attributed to the fact that
affinity to AT2 receptor in porcine membrane may not
be translated to the rat AT2 receptor.
N-Eth yloxycar bon yl-3-[4-(2-eth yl-5,7-dim eth ylim idazo-
[4,5-b]p yr id in -3-ylm eth yl)p h en yl]-5-isobu tylth iop h en e-
2-su lfon a m id e (4). Compound 3 was used according to the
general procedure and reacted with ethyl chloroformate (198
µL, 2.10 mmol), which after purification (CHCl3: MeOH 40:1)
1
gave compound 4 in 85% yield (48 mg, 0.087 mmol). H NMR
(CD3OD), δ: 7.52 (d, J ) 8.1 Hz, 2H), 7.16 (d, J ) 8.1 Hz, 2H),
6.99 (s, 1H), 6.78 (s, 1H), 5.55 (s, 2H), 3.92 (q, J ) 7.1 Hz,
2H), 2.87 (q, J ) 7.6 Hz, 2H), 2.65 (d, J ) 7.1 Hz, 2H), 2.60 (s,
3H), 2.56 (s, 3H), 1.86 (m, 1H), 1.29 (t, J ) 7.6 Hz, 3H), 1.07
(t, J ) 7.1 Hz, 3H), 0.95 (d, J ) 6.6 Hz, 6H); 13C NMR (CD3-
OD), δ: 158.2, 156.0, 154.2, 150.5, 148.4, 145.5, 139.7, 138.2,
135.6, 135.1, 133.0, 130.9, 127.7, 120.8, 63.1, 46.0, 40.0, 31.9,
24.1, 22.6, 22.2, 16.5, 14.8, 12.3; Anal. (C28H34N4O4S2) C, H,
N.
Con clu sion
In summary, the data presented demonstrate that the
first nonpeptide AT1 receptor agonist disclosed, the
thiophene derivative 1, also acts as an agonist at the
AT2 receptor type. Furthermore the finding that 12, in
contrast to 1, does not promote an effect in the secretor
model in vivo may suggest that the agonistic effects are
not likely to be retained if the imidazopyridine in the
upper part of 1 is replaced with a quinazolinone system,
a modification which was made in an attempt to
improve the AT2/AT1 ratio with respect to binding
affinities. Small structural modifications within the two
series examined had, in general, a considerably larger
impact on the binding affinity to the AT2 receptor than
to the AT1 receptor subtype. It is our belief that the
prototype compound 1 (possibly in combination with a
properly selected AT1 receptor antagonist) may provide
a useful research tool for studies of the role of the AT2
receptor in vivo. This substance is, to the best of our
knowledge, the first nonpeptidic low-molecular weight
compound demonstrated to exert an agonistic effect
mediated through the AT2 receptor.
N-Isobu tyloxyca r bon yl-3-[4-(2-eth yl-5,7-d im eth ylim i-
d a z o [4,5-b ]p y r i d i n -3-y lm e t h y l)p h e n y l]-5-i s o b u t y l-
th iop h en e-2-su lfon a m id e (5). Compound 3 was used ac-
cording to the general procedure and reacted with iso-butyl
chloroformate (269 µL, 2.10 mmol), which after purification
(CHCl3: MeOH 40:1) gave compound 5 in 85% yield (46 mg,
1
0.079 mmol). H NMR (CD3OD), δ: 7.50 (d, J ) 8.2 Hz, 2H),
7.17 (d, J ) 8.2 Hz, 2H), 7.00 (s, 1H), 6.81 (s, 1H), 5.56 (s,
2H), 3.69 (d, J ) 6.5 Hz, 2H), 2.87 (q, J ) 7.6 Hz, 2H), 2.68 (d,
J ) 7.1 Hz, 2H), 2.60 (s, 3H), 2.57 (s, 3H), 1.90 (m, 1H), 1.73
(m, 1H), 1.30 (t, J ) 7.5 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 6H),
0.78 (d, J ) 6.7 Hz, 6H); 13C NMR (CD3OD), δ: 158.2, 154.9,
154.2, 151.1, 148.5, 145.9, 139.7, 138.3, 135.5, 134.6, 133.1,
130.9, 127.8, 120.9, 73.3, 46.0, 40.1, 31.9, 29.1, 24.1, 22.7, 22.3,
19.4, 16.5, 12.3; Anal. (C30H38N4O4S2) C, H, N.
N-Hexyloxycar bon yl-3-[4-(2-eth yl-5,7-dim eth ylim idazo-
[4,5-b]p yr id in -3-ylm eth yl)p h en yl]-5-isobu tylth iop h en e-
2-su lfon a m id e (6). Compound 3 was used according to the
general procedure and reacted with hexyl chloroformate (339
µL, 2.10 mmol), which after purification (CHCl3: MeOH 40:1)
1
gave compound 6 in 74% yield (47 mg, 0.077 mmol). H NMR
(CD3OD), δ: 7.45 (d, J ) 8.3 Hz, 2H), 7.17 (d, J ) 8.3 Hz, 2H),
7.00 (s, 1H), 6.81 (s, 1H), 5.57 (s, 2H), 3.91 (t, J ) 6.4 Hz, 2H),
2.87 (q, J ) 7.6 Hz, 2H), 2.67 (d, J ) 7.1 Hz, 2H), 2.60 (s, 3H),
2.56 (s, 3H), 1.88 (m, 1H), 1.45-1.02 (m, 11H), 0.95 (d, J )
6.6 Hz, 6H), 0.85 (t, J ) 6.6 Hz, 3H); 13C NMR (CD3OD), δ:
158.2, 154.2, 153.3, 151.8, 148.4, 146.5, 139.7, 138.4, 135.2,
133.6, 133.0, 130.8, 127.8, 120.9, 67.4, 46.0, 40.1, 32.6, 31.9,
29.8, 26.5, 23.7, 22.7, 22.3, 16.5, 14.5, 12.3; Anal. (C32H42N4O4S2)
C, H, N.
N-(3-Cyclop en t a n ylp r op ion yl)-3-[4-(2-et h yl-5,7-d im e-
t h ylim id a zo[4,5-b]p yr id in -3-ylm et h yl)p h en yl]-5-isob u -
tylth iop h en e-2-su lfon a m id e (7). Compound 3 was used
according to the general procedure and reacted with 3-cyclo-
pentanylpropionyl chloride (317 µL, 2.10 mmol), which after
Exp er im en ta l Section
Ch em istr y. Gen er a l Con sid er a tion s. 1H and 13C NMR
spectra were recorded on a J EOL J NM-EX 270 spectrometer
at 270.2 and 67.8 MHz, respectively. Chemical shifts are given
as δ values (ppm) downfield from tetramethylsilane. Infrared
spectra were recorded on a Perkin-Elmer Model 1605 FT-IR
and are reported as λmax (cm-1). For neat solids the instrument
was equipped with a Microfocus Beam Condenser with ZnSe
lenses in
a Diasqueeze Pulse Diamond Compressor Cell
(Graseby Specac Inc., Woodstock, GA). Elemental analyses
were performed by Mikro Kemi AB, Uppsala, Sweden or
Analytische Laboratorien, Lindlar, Germany. Flash column

AT2 Receptor Agonist in Vivo
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1541
purification (CHCl3: MeOH 40:1) gave compound 7 in 76%
yield (48 mg, 0.079 mmol). H NMR (CD3OD), δ: 7.48 (d, J )
(10% aq) and water. The organic phase was dried, evaporated
and purified by column chromatography (hexane:acetone 3:1)
to give the pure compound 11 in 79% yield (21.3 mg, 0.033
mmol). 1H NMR (CDCl3), δ: 9.13 (d, J ) 2.6 Hz, 1H), 8.51
(dd, J ) 3.0, 2.6 Hz, 1H), 7.77 (d, J ) 9.1 Hz, 1H), 7.44 (d, J
) 8.3 Hz, 2H), 7.24 (d, J ) 8.3 Hz, 2H), 6.73 (s, 1H), 5.43 (s,
2H), 4.04 (t, J ) 6.6 Hz, 2H), 2.80 (t, J ) 7.4 Hz, 2H), 2.69 (d,
J ) 7.1 Hz, 2H), 2.00-1.90 (m, 3H), 1.54-1.43 (m, 2H), 1.30-
1.19 (m, 2H), 1.04 (t, J ) 7.4 Hz, 3H), 0.98 (d, J ) 6.6 Hz,
6H), 0.85 (t, J ) 7.3 Hz, 3H); 13C NMR (CDCl3), δ: 161.5, 160.7,
151.8, 151.3, 150.0, 145.8, 145.4, 135.9, 133.8, 129.6, 129.4,
128.7, 128.4, 126.5, 123.8, 120.3, 66.9, 46.6, 39.3, 37.2, 30.5,
1
8.2 Hz, 2H), 7.19 (d, J ) 8.2 Hz, 2H), 7.00 (s, 1H), 6.82 (s,
1H), 5.58 (s, 2H), 2.89 (q, J ) 7.5 Hz, 2H), 2.69 (d, J ) 7.0 Hz,
2H), 2.60 (s, 3H), 2.57 (s, 3H), 1.92 (m, 1H), 1.89 (t, J ) 7.5
Hz, 3H), 1.66-1.35 (m, 8H), 1.35-1.22 (m, 4H), 0.95 (d, J )
6.6 Hz, 6H), 0.87 (m, 1H); 13C NMR (CD3OD), δ: 174.2, 158.1,
154.2, 152.0, 148.5, 146.3, 139.8, 138.6, 135.3, 133.6, 133.2,
130.9, 130.8, 127.9, 120.9, 46.0, 40.8, 40.1, 36.3, 33.5, 31.9, 31.8,
26.1, 24.1, 22.6, 22.3, 16.5, 12.3; Anal. (C33H42N4O3S2) C,
H, N.
N-(Dip h en yla cetyl)-3-[4-(2-eth yl-5,7-d im eth ylim id a zo-
[4,5-b]p yr id in -3-ylm eth yl)p h en yl]-5-isobu tylth iop h en e-
2-su lfon a m id e (8). Compound 3 was used according to the
general procedure and reacted with diphenylacetyl chloride
(485 mg, 2.10 mmol), which after purification (CHCl3: MeOH
30.4, 22.2, 20.3, 18.7, 13.8, 13.5; IR (compression cell), cm-1
:
3522, 3098, 2963, 2871, 1745, 1679, 1617, 1567, 1524; Anal.
(C31H36N4O7S2) C, H, N.
5-Isobu tyl-3-[4-(6-a m in o-4-oxo-2-p r op yl-4H-qu in a zolin -
3-ylm e t h yl)p h e n yl]-N -t er t -b u t ylt h iop h e n e -2-su lfon a -
m id e (22). Compound 10 (428 mg, 0.717 mmol) and Pd/C
(10%, 43.2 mg) were dissolved in MeOH (10 mL), and HCO2-
NH4 (230 mg, 2.98 mmol) was added under N2 (g) atmosphere.
After an additional 30 min, HCO2NH4 (55.3 mg) was added,
and after 1 h a third portion of HCO2NH4 (55.3 mg) was added.
The solution was stirred for 1 h and thereafter diluted with
EtOAc (150 mL). The organic phase was washed with water
and brine, dried, and concentrated under vacuum. The crude
product was purified by column chromatography (hexane:
acetone 2:1) to afford the entitled product in 92% yield (375
mg, 0.659 mmol). 1H NMR (10%DMSO-d6 in CDCl3), δ: 7.47-
7.39 (m, 4H), 7.12-7.00 (m, 3H), 6.62 (s, 1H), 5.31 (s, 2H), 4.77
(brs, 2H), 2.64-2.55 (m, 4H), 2.06 (s, 1H), 1.77-1.67 (m, 3H),
0.91-0.84 (m, 18H); 13C NMR (10%DMSO-d6 in CDCl3), δ:
161.6, 153.6, 153.3, 147.9, 145.3, 142.2, 136.2, 136.0, 133.9,
129.3, 128.7, 127.0, 126.0, 123.4, 120.6, 109.0, 54.1, 46.0, 36.0,
1
40:1) gave compound 8 in 74% yield (50 mg, 0.074 mmol). H
NMR (CDCl3), δ: 7.8-7.2 (m, 15H), 6.96 (s, 1H), 5.77 (s, 2H),
3.18 (q, J ) 7.5 Hz, 2H), 3.05-2.95 (m, 5H), 2.92 (s, 3H), 2.25
(m, 1H), 1.66 (t, J ) 7.5 Hz, 3H), 1.30 (d, J ) 6.6 Hz, 6H); 13
C
NMR (CDCl3), δ: 155.9, 152.2, 151.6, 147.2, 145.9, 139.4, 138.4,
137.4, 136.8, 133.2, 132.3, 129.3, 128.9, 128.6, 128.5, 128.4,
127.5, 126.2, 119.5, 58.2, 44.9, 39.3, 30.4, 23.9, 22.2, 21.4, 18.2,
16.3, 12.0; Anal. (C39H40N4O3S2) C, H, N.
N-P en ta n oyl-3-[4-(2-eth yl-5,7-d im eth ylim id a zo[4,5-b]-
p yr id in -3-ylm et h yl)p h en yl]-5-isob u t ylt h iop h en e-2-su l-
fon a m id e (9). Compound 3 was used according to the general
procedure and reacted with valeryl chloride (249 µL, 2.10
mmol), which after purification (CHCl3: MeOH 40:1) gave
compound 9 in 79% yield (45 mg, 0.079 mmol). 1H NMR (CD3-
OD), δ: 7.46 (d, J ) 8.3 Hz, 2H), 7.20 (d, J ) 8.3 Hz, 2H), 7.02
(s, 1H), 6.82 (s, 1H), 5.58 (s, 2H), 2.91 (q, J ) 7.6 Hz, 2H),
2.70 (d, J ) 7.1 Hz, 2H), 2.60 (s, 3H), 2.57 (s, 3H), 1.90 (m,
1H), 1.83 (t, J ) 7.6 Hz, 2H), 1.32 (t, J ) 7.6 Hz, 3H), 1.27 (m,
2H), 1.08 (m, 2H), 0.96 (d, J ) 6.6 Hz, 6H), 0.75 (t, J ) 7.1
Hz, 3H); 13C NMR (CD3OD), δ: 173.4, 158.1, 154.3, 152.2,
148.4, 146.5, 139.8, 138.6, 135.3, 133.2, 133.1, 130.9, 130.8,
127.9, 120.9, 46.0, 40.1, 36.4, 31.9, 27.6, 24.1, 23.2, 22.6, 22.3,
16.5, 14.2, 12.3. Anal. (C30H38N4O3S2×0.5H2O) C, H, N.
30.6, 30.1, 29.2, 21.8, 20.6, 13.6; IR (compression cell), cm-1
:
3315, 2961, 2859, 1657, 1591; Anal. (C30H38N4O3S2) C, H, N.
5-Isob u t yl-3-{4-[6-(a ce t yla m in o)-4-oxo-2-p r op yl-4H -
qu in a zolin -3-ylm eth yl]p h en yl}-N-ter t-bu tylth iop h en e-2-
su lfon a m id e (23). Compound 22 (30 mg, 0.053 mmol) and
DIEA (27 mL, 0.16 mmol) were dissolved in CH2Cl2 (5 mL)
under N2 (g) atmosphere and cooled on an ice bath. Acetyl
chloride (7.5 µL, 0.11 mmol) was added to the stirred solution.
The reaction mixture was stirred overnight at ambient tem-
perature. CHCl3 (50 mL) was added, and the organic phase
was washed with NaHCO3 (sat.), dried, and evaporated under
vacuum. The crude product was purified by column chroma-
tography (isohexane: EtOAc 10:1) to obtain the entitled
compound in 93% yield (30 mg, 0.049 mmol). 1H NMR (CDCl3),
δ: 8.54 (s, 1H), 8.29 (dd, J ) 8.9, 2.3 Hz, 1H), 8.15 (d, J ) 2.3
Hz, 1H), 7.62 (d, J ) 8.9 Hz, 1H), 7.55 (d, J ) 8.3 Hz, 2H),
7.19 (d, J ) 2.2 Hz, 2H), 6.70 (s, 1H), 5.40 (s, 2H), 4.46 (s,
1H), 2.80-2.63 (m, 4H), 2.12 (s, 3H), 1.90-1.76 (m, 3H), 1.24
(s, 1H), 0.98-0.85 (m, 18H); 13C NMR (CDCl3), δ: 168.9, 162.3,
155.6, 148.5, 143.5, 142.5, 137.1, 136.3, 136.2, 134.3, 129.6,
128.9, 127.7, 127.3, 126.3, 120.2, 115.9, 54.5, 46.2, 39.1, 36.8,
5-Isobu tyl-3-[4-(6-n itr o-4-oxo-2-p r op yl-4H-qu in a zolin -
3-ylm e t h yl)p h e n yl]-N -t er t -b u t ylt h iop h e n e -2-su lfon a -
m id e (10). 3-(4-Bromo-benzyl)-6-nitro-2-propyl-3H-quinazolin-
4-one (672 mg, 1.67 mmol), 2 (789 mg, 2.50 mmol), Pd(PPh3)4
(60 mg, 0.052 mmol), and NaOH (10 mL, 1M) were dissolved
in toluene (40 mL) and ethanol (6 mL) under N2 (g) atmo-
sphere. The reaction mixture was heated to 100 °C and stirred
for 2 h. After dilution with water (50 mL), the reaction mixture
was extracted with EtOAc and washed with brine and water.
The organic phase was dried and concentrated under vacuum.
The crude product was purified by column chromatography
(hexane:acetone 5:1) to give the entitled product in 82% yield
(814 mg, 1.36 mmol). 1H NMR (CDCl3), δ: 9.15 (d, J ) 2.6 Hz,
1H), 8.54 (dd, J ) 8.9, 2.6 Hz, 1H), 7.80 (d, J ) 8.9 Hz, 1H),
7.59 (d, J ) 8.3 Hz, 2H), 7.26 (d, J ) 8.1 Hz, 2H), 6.72 (s, 1H),
5.45 (s, 2H), 4.23 (s, 1H), 2.78 (t, J ) 7.4 Hz, 2H), 2.66 (d, J )
7.1 Hz, 2H), 1.95-1.79 (m, 3H), 1.05-0.90 (m, 18H); 13C NMR
(CDCl3), δ: 161.4, 160.7, 151.1, 148.6, 145.4, 142.3, 136.7,
135.5, 134.6, 129.7, 128.8, 128.6, 128.4, 126.5, 123.8, 120.3,
54.5, 46.5, 39.1, 37.1, 30.5, 29.4, 22.1, 20.2, 13.8; IR (compres-
sion cell), cm-1: 3293, 3079, 2964, 2869, 1685, 1573; Anal.
(C30H36N4O5S2) C, H, N.
N-Bu t yloxyca r b on yl-5-isob u t yl3-[4-(6-n it r o-4-oxo-2-
p r op yl-4H -q u in a zolin -3-ylm et h yl)p h en yl]t h iop h en e-2-
su lfon a m id e (11). Compound 10 (25 mg, 0.042 mmol) and
anisole (50 µL) were dissolved in TFA (1 mL) and stirred
overnight. The reaction mixture was evaporated under vacuum
and coevaporated with acetonitrile twice. The residue was
dissolved in pyridine (1 mL), and pyrrolidinopyridine (6 mg,
0.04 mmol) was added. The solution was cooled on an ice bath,
and butyl chloroformate (107 µL, 0.84 mmol) was added under
a N2 (g) atmosphere. The reaction was stirred at ambient
temperature overnight. The solvent was removed under vacuum
and coevaporated with acetonitrile twice. The residue was
dissolved in ethyl acetate (50 mL) and washed with citric acid
30.4, 29.4, 24.4, 22.1, 20.5, 13.8; IR (compression cell), cm-1
:
3301, 2965, 2870, 1658, 1593, 1537; Anal. (C32H40N4O4S2) C,
H, N.
N-Bu t yloxyca r b on yl-5-isob u t yl3-{4-[6-(a cet yla m in o)-
4-o x o -2-p r o p y l-4H -q u in a zo lin -3-y lm e t h y l]p h e n y l}-
th iop h en e-2-su lfon a m id e (12). Compound 23 (30 mg, 0.049
mmol) and anisole (50 µL) were dissolved in TFA (1 mL) and
stirred overnight. The reaction mixture was evaporated under
vacuum and coevaporated with acetonitrile twice. The residue
was dissolved in pyridine (1 mL), and pyrrolidinopyridine (7
mg, 0.05 mmol) was added. The solution was cooled on an ice
bath, and butyl chloroformate (60 µL, 0.47 mmol) was added
under a N2 (g) atmosphere. The reaction was stirred at
ambient temperature overnight. The solvent was removed
under vacuum and coevaporated with acetonitrile twice. The
residue was dissolved in ethyl acetate (50 mL) and washed
with citric acid (10% aq) and water. The organic phase was
dried, evaporated, and purified by column chromatography
(isohexane:EtOAc 9:1) to give the pure compound 12 in 71%
yield (22.7 mg, 0.034 mmol). 1H NMR (CDCl3), δ: 9.11 (s, 1H),

1542 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wan et al.
8.47 (brs, 1H), 8.17 (s, 1H), 7.61 (d, J ) 8.9 Hz, 1H), 7.48 (d,
J ) 8.3 Hz, 2H), 7.08 (d, J ) 8.1 Hz, 2H), 6.72 (s, 1H), 5.45 (s,
2H), 4.04 (t, J ) 6.6 Hz, 2H), 2.70-2.63 (m, 4H), 1.91 (s, 3H),
1.88-1.70 (m, 2H), 1.53-1.40 (m, 2H), 1.30-1.15 (m, 2H),
0.99-0.80 (m, 12H); 13C NMR (CDCl3), δ: 170.3, 162.6, 162.5,
156.2, 151.1, 151.0, 144.8, 137.3, 135.7, 134.2, 131.8, 129.9,
129.6, 128.2, 127.4, 125.7, 119.5, 116.1, 66.4, 46.5, 39.3, 36.2,
30.5, 23.8, 22.3, 20.7, 18.8, 13.8, 13.6; IR (compression cell),
1.77 (m, 3H), 1.01-0.95 (m, 18H);13C NMR (CDCl3), δ: 162.3,
160.2, 155.6, 148.4, 143.6, 142.5, 139.3, 136.9, 136.3, 136.1,
134.2, 131.2, 129.5, 129.0, 128.7, 127.8, 127.6, 126.1, 120.1,
116.6, 54.4, 46.1, 39.1, 36.6, 30.4, 29.4, 22.1, 20.4, 13.8; Anal.
(C35H40N4O4S2) C, H, N.
N-Bu tyloxyca r bon yl-5-isobu tyl-3-(4-{6-[(th iop h en e-2-
c a r b o n y l)-a m in o ]-4-o x o -2-p r o p y l-4H -q u in a zo lin -3-
ylm eth yl}p h en yl)-th iop h en e-2-su lfon a m id e (14). Com-
pound 25 (23 mg, 0.034 mmol) and anisole (50 µL) were
dissolved in TFA (1 mL) and stirred overnight. The reaction
mixture was evaporated under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in pyridine (1
mL), and pyrrolidinopyridine (7 mg, 0.05 mmol) was added.
The solution was cooled on an ice bath, and butyl chlorofor-
mate (60 µL, 0.47 mmol) was added under a N2 (g) atmosphere.
The reaction was stirred at ambient temperature overnight.
The solvent was removed under vacuum and coevaporated
with acetonitrile twice. The residue was dissolved in ethyl
acetate (50 mL) and washed with citric acid (10% aq) and
water. The organic phase was dried, evaporated, and purified
by column chromatography (isohexane:EtOAc 9:1) to give the
pure compound 14 in 89% yield (22 mg, 0.030 mmol). 1H NMR
(CDCl3), δ: 9.70 (brs, 1H), 9.54 (s, 1H), 8.75 (m, 1H), 8.41 (m,
1H), 7.86 (m, 1H), 7.64 (d, J ) 8.9 Hz, 1H), 7.51 (d, J ) 4.8
Hz, 1H), 7.35 (d, J ) 8.1 Hz, 2H), 7.04 (brs, 1H), 6.76-6.70
(m, 3H), 5.15 (brs, 2H), 4.10 (t, J ) 6.7 Hz, 2H), 2.68 (d, J )
7.0 Hz, 2H), 2.53 (m, 2H), 1.93 (sep, J ) 6.7 Hz, 1H), 1.69
(sxt, J ) 7.4 Hz, 2H), 1.53 (m, 2H), 1.25 (m, 2H), 0.97 (d, J )
6.6 Hz, 6H), 0.91-0.96 (m, 6H); 13C NMR (CDCl3), δ: 162.7,
160.2, 155.6, 151.0, 150.9, 145.2, 138.8, 137.0, 135.7, 133.4,
131.3, 129.9, 129.7, 129.6, 128.3, 127.7, 125.3, 119.6, 116.9,
66.5, 46.1, 39.2, 36.3, 30.5, 30.5, 22.2, 20.4, 18.8, 13.7, 13.6;
Anal. (C36H40N4O6S3) C, H, N.
5-Isob u t yl-3-{4-[6-(e t h yla m in o)-4-oxo-2-p r op yl-4H -
qu in a zolin -3-ylm eth yl]p h en yl}-N-ter t-bu tylth iop h en e-2-
su lfon a m id e (26). Compound 22 (200 mg, 0.353 mmol),
acetaldehyde (24 µL, 0.42 mmol), and acetic acid (101 µL, 1.76
mmol) were dissolved in CH2ClCH2Cl (20 mL) and stirred for
30 min. NaB(OAc)3H (172 mg, 0.811 mmol) was added, and
the reaction mixture was stirred overnight. CHCl3 (50 mL) was
added, and the organic phase was washed with NaHCO3 and
dried. The solvent was removed under vacuum, and the crude
product was purified by column chromatography (isohexane:
EtOAc 10:1) to give the entitled product in 95% yield (200 mg,
0.336 mmol). 1H NMR (CDCl3), δ: 7.60-7.53 (m, 3H), 7.29 (d,
J ) 2.8 Hz, 1H), 7.24 (d, J ) 9.6 Hz, 2H), 7.05 (dd, J ) 2.8,
8.8 Hz, 1H), 6.71 (s, 1H), 5.43 (s, 2H), 4.14 (s, 1H), 3.23 (q, J
) 7.3 Hz, 2H), 2.73-2.64 (m, 4H), 1.31-1.77 (m, 3H), 1.30 (t,
J ) 7.1 Hz, 3H), 1.00 (t, J ) 7.3 Hz, 3H), 0.95-0.93 (m, 15H);
13C NMR (CDCl3), δ: 162.3, 152.9, 148.4, 147.2, 142.5, 137.5,
136.8, 136.4, 134.2, 129.5, 128.8, 127.5, 126.5, 122.5, 121.2,
104.9, 54.5, 46.2, 39.1, 38.5, 36.6, 30.5, 29.5, 22.1, 21.0, 14.6,
13.9; IR (compression cell), cm-1: 3380, 3292, 2965, 2930, 2871,
1657, 1648, 1591, 1512; Anal. (C32H42N4O3S2) C, H, N.
cm-1
: 3304, 2960, 2931, 2871, 1749, 1671, 1593; Anal.
(C33H40N4O6S2) C, H, N.
5-Isobu t yl-3-{4-[6-(b en zoyla m in o)-4-oxo-2-p r op yl-4H -
qu in a zolin -3-ylm eth yl]p h en yl}-N-ter t-bu tylth iop h en e-2-
su lfon a m id e (24). Compound 22 (30 mg, 0.053 mmol) and
DIEA (27 mL, 0.16 mmol) were dissolved in CH2Cl2 (5 mL)
under N2 (g) atmosphere and cooled on an ice bath. Benzoyl
chloride (12 µL, 0.11 mmol) was added to the stirred solution.
The reaction mixture was stirred overnight at ambient tem-
perature. CHCl3 (50 mL) was added, and the organic phase
was washed with NaHCO3 (sat.), dried, and evaporated under
vacuum. The crude product was purified by column chroma-
tography (isohexane:EtOAc 10:1) to obtain the entitled com-
1
pound in 92% yield (33 mg, 0.049 mmol). H NMR (CDCl3), δ:
8.83 (brs, 1H), 8.50 (d, J ) 8.7 Hz, 1H), 8.34 (s, 1H), 7.91 (d,
J ) 7.2 Hz, 2H), 7.68 (d, J ) 8.8 Hz, 1H), 7.54-7.41 (m, 5H),
7.07 (m, 2H), 6.71 (s, 1H), 5.30 (brs, 2H), 4.33 (s, 1H), 2.68-
2.63 (m, 4H), 1.95-1.76 (m, 3H), 1.24 (s, 1H), 1.01-0.90 (m,
18H); 13C NMR (CDCl3), δ: 166.0, 162.2, 155.9, 148.4, 142.4,
137.4, 136.3, 136.0, 134.4, 134.2, 131.9, 129.5, 128.9, 128.6,
127.5, 127.4, 126.2, 120.0, 116.7, 54.5, 46.1, 39.1, 36.5, 30.4,
29.4, 22.1, 20.5, 13.8; Anal. (C37H44N4O3S2) C, H, N.
N-Bu tyloxycar bon yl-5-isobu tyl-3-{4-[6-(ben zoylam in o)-
4-o x o -2-p r o p y l-4H -q u in a zo lin -3-y lm e t h y l]p h e n y l}-
th iop h en e-2-su lfon a m id e (13). Compound 24 (33 mg, 0.049
mmol) and anisole (50 µL) were dissolved in TFA (1 mL) and
stirred overnight. The reaction mixture was evaporated under
vacuum and coevaporated with acetonitrile twice. The residue
was dissolved in pyridine (1 mL), and pyrolidinopyridine (7
mg, 0.05 mmol) was added. The solution was cooled on an ice
bath, and butyl chloroformate (60 µL, 0.47 mmol) was added
under a N2 (g) atmosphere. The reaction was stirred at
ambient temperature overnight. The solvent was removed
under vacuum and coevaporated with acetonitrile twice. The
residue was dissolved in ethyl acetate (50 mL) and washed
with citric acid (10% aq) and water. The organic phase was
dried, evaporated and purified by column chromatography
(isohexane:EtOAc 9:1) to give the pure compound 13 in 70%
yield (25 mg, 0.034 mmol). 1H NMR (CDCl3), δ: 9.87 (brs, 1H),
9.76 (s, 1H), 8.90 (d, J ) 8.3 Hz, 1H), 8.47 (d, J ) 2.2 Hz, 1H),
7.91 (d, J ) 7.3 Hz, 2H), 7.68 (d, J ) 9.0 Hz, 1H), 7.56 (m,
1H), 7.47-7.41 (m, 2H), 7.35 (d, J ) 8.2 Hz, 2H), 6.73-6.69
(m, 3H), 4.91 (brs, 2H), 4.10 (t, J ) 6.7 Hz, 2H) 2.68 (d, J )
7.0 Hz, 2H), 2.51 (m, 2H), 1.93 (sep, J ) 6.7 Hz, 1H), 1.69
(sxt, J ) 7.4 Hz, 2H), 1.52 (m, 2H), 1.23 (m, 2H), 0.97 (d, J )
6.6 Hz, 6H), 0.90-0.79 (m, 6H); 13C NMR (CDCl3), δ: 165.9,
162.6, 155.5, 151.0, 150.9, 145.1, 137.5, 135.8, 133.9, 133.6,
132.1, 131.6, 129.9, 129.7, 128.3, 128.1, 128.0, 125.7, 119.6,
116.8, 66.5, 45.8, 39.2, 36.3, 30.5, 30.5, 22.2, 20.4, 18.7, 13.7,
13.6; Anal. (C38H42N4O6S2) C, H, N.
5-Isob u t yl-3-{4-[6-(b en zyla m in o)-4-oxo-2-p r op yl-4H -
qu in a zolin -3-ylm eth yl]p h en yl}-N-ter t-bu tylth iop h en e-2-
su lfon a m id e (27). Compound 22 (200 mg, 0.353 mmol),
benzaldehyde (39 µL, 0.39 mmol), and acetic acid (101 µL, 1.76
mmol) were dissolved in CH2ClCH2Cl (20 mL) and stirred for
30 min. NaB(OAc)3H (172 mg, 0.811 mmol) was added, and
the reaction mixture was stirred overnight. CHCl3 (50 mL) was
added, and the organic phase was washed with NaHCO3 and
dried. The solvent was removed under vacuum, and the crude
product was purified by column chromatography (isohexane:
EtOAc 10:1) to give the entitled product in 95% yield (220 mg,
5-Isob u t yl-3-(4-{6-[(t h iop h en e-2-ca r b on yl)-a m in o]-4-
oxo-2-p r op yl-4H-q u in a zolin -3-ylm et h yl}p h en yl)-N-ter t-
bu tylth iop h en e-2-su lfon a m id e (25). Compound 22 (30 mg,
0.053 mmol) and DIEA (27 mL, 0.16 mmol) were dissolved in
CH2Cl2 (5 mL) under N2 (g) atmosphere and cooled on an ice
bath. Thiophene-2-carbonyl chloride (12 µL, 0.11 mmol) was
added to the stirred solution. The reaction mixture was stirred
overnight at ambient temperature. CHCl3 (50 mL) was added,
and the organic phase was washed with NaHCO3 (sat.), dried,
and evaporated under vacuum. The crude product was purified
by column chromatography (isohexane:EtOAc 10:1) to obtain
1
0.335 mmol). H NMR (CDCl3), δ: 7.62-7.55 (m, 3H), 7.41-
7.28 (m, 6H), 7.26-7.21 (m, 2H), 7.09 (dd, J ) 8.9, 2.9 Hz,
1H), 6.71 (s, 1H), 5.41 (s, 2H), 4.42 (s, 2H), 4.10 (s, 1H), 2.75-
2.62 (m, 4H), 1.95-1.74 (m 3H), 1.03-0.94 (m, 18H); 13C NMR
(CDCl3), δ: 148.4, 146.9, 142.4, 136.6, 136.4, 134.2, 129.5,
128.7, 127.5, 126.4, 122.3, 121.1, 105.3, 54.4, 48.1, 46.2, 39.1,
36.5, 30.4, 29.6, 29.4, 22.1, 20.9, 13.8; IR (compression cell),
cm-1: 3294, 2960, 1655, 1619, 1509; Anal. (C37H44N4O3S2) C,
H, N.
1
the entitled compound in 65% yield (23 mg, 0.034 mmol). H
NMR (CDCl3), δ: 8.56 (brs, 1H), 8.44 (dd, J ) 8.9, 2.5 Hz, 1H),
8.26 (d, J ) 2.3 Hz, 1H), 7.75 (d, J ) 3.6 Hz, 1H), 7.69 (d, J )
8.7 Hz, 1H), 7.55-7.52 (m, 3H), 7.14-7.06 (m, 3H), 6.72 (s,
1H), 5.37 (brs, 2H), 4.32 (s, 1H), 2.68-2.63 (m, 4H), 1.96-

AT2 Receptor Agonist in Vivo
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1543
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
28-30. Compound 26 (67 mg, 0.11 mmol) and DIEA (97 µL,
0.56 mmol) were dissolved in CH2Cl2 (5 mL) and cooled on an
ice bath. The acid chloride (5 equiv) was added to the solution,
and the reaction mixture was stirred overnight at ambient
temperature. CHCl3 (50 mL) was added, and the organic phase
was washed with NaHCO3 (sat.), dried, and concentrated
under vacuum. The crude products were purified by column
chromatography to give the pure products 28-30.
5-Isobu tyl-3-{4-[6-(N-acetyleth ylam in o)-4-oxo-2-pr opyl-
4H-qu in azolin -3-ylm eth yl]ph en yl}-N-ter t-bu tylth ioph en e-
2-su lfon a m id e (28). Compound 26 was used according to the
general procedure and reacted with acetyl chloride (40 µL, 0.56
mmol) which gave after purification (isohexane:EtOAc 10:1)
compound 28 in 92% yield (66 mg, 0.10 mmol). 1H NMR
(CDCl3), δ: 8.10 (d, J ) 2.3 Hz, 1H), 7.81 (d, J ) 8.4 Hz, 1H),
7.55 (m, 1H), 7.45 (d, J ) 8.3 Hz, 2H), 7.25 (d, J ) 8.1 Hz,
2H), 6.73 (s, 1H), 5.45 (s, 2H), 4.05 (t, J ) 6.6 Hz, 2H), 3.81 (q,
J ) 7.1 Hz, 2H), 2.83 (t, J ) 7.4 Hz, 2H), 2.70 (d, J ) 7.1 Hz,
2H), 1.94-1.85(m, 6H), 1.56-1.44 (m, 1H), 1.28-1.19 (m, 2H),
1.16-1.06 (m, 6H), 0.98 (d, J ) 6.6 Hz, 6H), 0.85 (t, J ) 7.42
Hz, 3H); 13C NMR (CDCl3), δ: 169.8, 161.1, 159.5, 151.7, 150.2,
145.8, 144.0, 141.7, 135.6, 135.2, 133.9, 130.8, 129.7, 129.3,
127.5, 126.4, 126.2, 120.5, 66.9, 46.8, 44.1, 39.3, 36.2, 30.5, 30.4,
22.9, 22.2, 21.1, 18.7, 13.8, 13.6, 13.1; IR (compression cell),
cm-1: 2960, 2871, 1746, 1671, 1591; Anal. (C34H44N4O4S2) C,
H, N.
cording to the general procedure and reacted with acetyl
chloride (40 µL, 0.56 mmol), which after purification (isohex-
ane:EtOAc 10:1) gave compound 31 in 96% yield (75 mg, 0.11
1
mmol). H NMR (CDCl3), δ: 8.00 (s, 1H), 7.70-7.57 (m, 2H),
7.30-7.18 (m, 9H), 6.71 (s, 1H), 5.41 (brs, 2H), 4.96 (s, 2H),
4.06 (s, 1H), 2.75 (m, 2H), 2.66 (d, J ) 7.3 Hz, 2H), 1.95-1.76
(m, 6H), 1.05-0.94 (m, 18H); 13C NMR (CDCl3), δ: 148.5,
142.3, 141.2, 136.8, 136.5, 135.8, 134.8, 134.6, 129.6, 128.8,
128.5, 128.4, 128.2, 127.5, 126.4, 126.1, 120.8, 54.5, 52.8, 46.4,
39.1, 36.6, 30.5, 29.5, 22.9, 22.1, 20.6, 13.8; IR (compression
cell), cm-1: 2959, 1672, 1653, 1592; Anal. (C39H46N4O4S2) C,
H, N.
5-Isobu tyl-3-{4-[6-(N-p en ta n oylben zyla m in o)-4-oxo-2-
pr opyl-4H-qu in azolin -3-ylm eth yl]ph en yl}-N-ter t-bu tylth -
iop h en e-2-su lfon a m id e (32). Compound 27 was used ac-
cording to the general procedure and reacted with pentanoyl
chloride (66 µL, 0.56 mmol), which after purification (isohex-
ane:EtOAc 10:1) gave compound 32 in 94% yield (78 mg, 0.11
1
mmol). H NMR (CDCl3), δ: 8.00 (d, J ) 2.2 Hz, 1H), 7.67-
7.54 (m, 3H), 7.33-7.15 (m, 8H), 6.72 (s, 1H), 5.42 (s, 2H), 4.96
(s, 2H), 4.10 (s, 1H), 2.73 (t, 2H, 7.5 Hz), 2.67 (d, J ) 7.1 Hz,
2H), 2.10 (br t, J ) 6.9 Hz, 2H), 1.90 (m, 1H), 1.82 (m, 2H),
1.60 (m, 2H), 1.22 (m, 2H), 1.01 (t, J ) 7.2 Hz, 3H), 0.98 (s,
9H), 0.96 (d, J ) 6.8 Hz, 6H), 0.81 (t, J ) 7.2 Hz, 3H); 13C
NMR (CDCl3), δ: 172.9, 161.8, 157.7, 148.6, 146.4, 142.4, 141.0,
140.8, 137.1, 136.4, 136.1, 135.0, 134.5, 129.7, 128.8, 128.7,
128.5, 127.5, 126.5, 121.0, 54.5, 53.1, 46.3, 39.1, 36.9, 34.3, 30.5,
29.5, 27.5, 22.3, 22.1, 20.5, 13.9; IR (compression cell), cm-1
:
5-Isobu tyl-3-{4-[6-(N-ben zoyl-eth yla m in o)-4-oxo-2-p r o-
p yl-4H -q u in a zolin -3-ylm et h yl]p h en yl}-N-ter t-b u t ylt h -
iop h en e-2-su lfon a m id e (29). Compound 26 was used ac-
cording to the general procedure and reacted with benzoyl
chloride (66 µL, 0.56 mmol), which after purification (isohex-
ane:EtOAc 10:1) gave compound 29 in 93% yield (73 mg, 0.10
3293, 3063, 2959, 1671, 1593; Anal. (C42H52N4O4S2) C, H, N.
5-Isobu tyl-3-{4-[6-(N-ben zoylben zylam in o)-4-oxo-2-pr o-
p yl-4H -q u in a zolin -3-ylm et h yl]p h en yl}-N-ter t-b u t ylt h -
iop h en e-2-su lfon a m id e (33). Compound 27 was used ac-
cording to the general procedure and reacted with benzoyl
chloride (65 µL, 0.56 mmol), which after purification (isohex-
ane:EtOAc 10:1) gave compound 33 in 93% yield (79 mg, 0.10
1
mmol). H NMR (CDCl3), δ: 8.04 (d, J ) 2.5 Hz, 1H), 7.56 (d,
J ) 8.9 Hz, 1H), 7.44 (d, J ) 8.3 Hz, 2H), 7.34-7.13 (m, 8H),
6.72 (s, 1H), 5.39 (s, 2H), 4.08-4.01 (m, 4H), 2.80-2.68 (m,
4H), 1.99-1.77 (m, 3H), 1.53-1.49 (m, 2H), 1.27-11.20 (m,
5H), 1.03-0.97 (m, 9H), 0.85 (t, J ) 7.3 Hz, 3H); 13C NMR
(CDCl3), δ: 170.3, 161.6, 158.5, 151.8, 150.0, 145.9, 142.1,
136.0, 135.7, 135.3, 135.1, 133.8, 130.7, 129.9, 129.6, 129.4,
128.7, 128.1, 127.0, 126.4, 124.5, 120.4, 66.9, 46.6, 45.7, 39.3,
36.5, 30.5, 30.4, 22.2, 20.9, 18.7, 13.9, 13.6, 13.1; IR (compres-
sion cell), cm-1: 3058, 2961, 2932, 2872, 1745, 1644; Anal.
(C39H46N4O4S2) C, H, N.
1
mmol). H NMR (CDCl3), δ: 8.04 (s, 1H), 7.57 (d, J ) 7.9 Hz,
2H), 7.49-7.35 (m, 3H), 7.34-7.11 (m, 11H), 6.71 (s, 1H), 5.37
(s, 2H), 5.21 (s, 2H), 4.19 (s, 1H), 2.72-2.64 (m, 4H), 1.94 (m,
1H), 1.77 (m, 2H), 1.00-0.94 (m, 18H); 13C NMR (CDCl3), δ:
170.6, 161.5, 157.5, 148.5, 142.3, 141.8, 136.8, 136.5, 135.9,
135.3,134.6, 134.4, 129.9, 129.5, 128.7, 128.5, 128.2, 127.9,
127.4, 126.4, 124.3, 120.4, 54.4, 53.9, 46.3, 39.1, 36.6, 30.4, 29.4,
22.0, 20.5, 13.8; IR (compression cell), cm-1: 3292, 2961, 1672,
1647; Anal. (C44H48N4O4S2) C, H, N.
5-Isobu tyl-3-(4-{6-[N-(th iop h en e-2-ca r bon yl)-eth yla m i-
n o]-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl}p h en yl)-N-
ter t-bu tylth iop h en e-2-su lfon a m id e (30). Compound 26 was
used according to the general procedure and reacted with
thiophene-2-carbonyl chloride (60 µL, 0.56 mmol) gave after
purification (isohexane:EtOAc 10:1) compound 30 in 92% yield
5-Isobu tyl-3-(4-{6-[N-(th ioph en e-2-car bon yl)-ben zylam i-
n o]-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl}p h en yl)-N-
ter t-bu tylth iop h en e-2-su lfon a m id e (34). Compound 27 was
used according to the general procedure and reacted with
thiophene-2-carbonyl chloride (60 µL, 0.56 mmol), which after
purification (isohexane:EtOAc 10:1) gave compound 34 in 86%
1
(73 mg, 0.10 mmol). H NMR (CDCl3), δ: 8.18 (d, J ) 2.3 Hz,
1
yield (73 mg, 0.095 mmol). H NMR (CDCl3), δ: 8.13 (d, J )
1H), 7.68 (d, J ) 8.6 Hz, 1H), 7.52 (dd, J ) 6.1, 2.5 Hz, 1H),
7.45 (d, J ) 8.1 Hz, 2H), 7.30-7.21 (m, 3H), 6.85 (d, J ) 2.6
Hz, 1H), 6.77 (m, 1H), 6.73 (s, 1H), 5.41 (s, 2H), 4.10-3.90
(m, 4H), 2.78-2.66 (m, 4H), 1.89-1.83 (m, 3H), 1.54-1.42 (m,
2H), 1.30-1.17 (m, 5H), 1.05-0.94 (m, 9H), 0.85 (t, J ) 7.3
Hz, 3H); 13C NMR (CDCl3), δ: 162.2, 162.0, 157.9, 151.6, 150.2,
146.7, 145.8, 140.8, 137.9, 136.4, 135.5, 133.6, 132.5, 130.8,
130.6, 129.5, 129.4, 128.7, 126.7, 126.4, 126.3, 121.0, 66.8, 46.5,
46.2, 39.3, 37.1, 30.5, 30.4, 22.2, 20.5, 18.7, 13.9, 13.6, 12.8;
IR (compression cell), cm-1:3074, 2961, 2872, 1747, 1672, 1591;
Anal. (C37H44N4O4S3 × H2O) C, H, N.
2.5 Hz, 1H), 7.64-7.50 (m, 3H), 7.36-7.18 (m, 9H), 6.89 (dd,
J ) 3.8, 1.1 Hz, 1H), 6.79 (dd, J ) 4.9, 3.8 Hz, 1H), 6.72 (s,
1H), 5.41 (s, 2H), 5.14 (s, 2H), 4.18 (s, 1H), 2.73 (t, J ) 7.6 Hz,
2H), 2.67 (d, J ) 6.9 Hz, 2H), 1.98-1.76 (m, 3H), 0.89-1.09
(m, 18H). 13C NMR (CDCl3), δ: 162.5, 161.7, 157.9, 148.5,
146.4, 142.4, 140.8, 137.5, 136.6, 136.4, 136.0, 135.6, 134.4,
132.8, 131.0, 129.6, 128.8, 128.7, 128.5, 128.3, 127.6, 126.7,
126.5, 126.3, 120.9, 54.7, 54.5, 46.4, 39.1, 36.9, 30.5, 29.4, 22.1,
20.4, 13.9; IR (compression cell), cm-1: 3289, 3081, 2963, 1672,
1624, 1609, 1589; Anal. (C42H46N4O4S3) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Com -
p ou n d s 31-34. Compound 27 (73 mg, 0.11 mmol) and DIEA
(97 µL, 0.56 mmol) were dissolved in CH2Cl2 (5 mL) and cooled
on an ice bath. The acid chloride (5 equiv) was added to the
solution, and the reaction mixture was stirred overnight at
ambient temperature. CHCl3 (50 mL) was added, and the
organic phase was washed with NaHCO3 (sat.), dried, and
concentrated under vacuum. The crude products were purified
by column chromatography to give the pure products 31-34.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
15-21. Compounds 28-34 and anisole (150 µL) was dissolved
in TFA (5 mL) and stirred overnight. The reaction mixture
was evaporated under vacuum and coevaporated with aceto-
nitrile twice. The residue was dissolved in pyridine (3 mL),
and pyrrolidinopyridine (1 equiv) was added. The solution was
cooled on an ice bath and the butyl chloroformate (20 equiv)
was added under a N2 (g) atmosphere. The reaction was stirred
at ambient temperature overnight. The solvent was removed
under vacuum and coevaporated with acetonitrile twice. The
residue was dissolved in ethyl acetate (50 mL) and washed
with citric acid (10% aq) and water. The organic phase was
5-Isobu tyl-3-{4-[6-(N-a cetyl-ben zyla m in o)-4-oxo-2-p r o-
p yl-4H -q u in a zolin -3-ylm et h yl]p h en yl}-N-ter t-b u t ylt h -
iop h en e-2-su lfon a m id e (31). Compound 27 was used ac-

1544 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wan et al.
dried, evaporated and purified by column chromatography to
give the pure compounds 15-21.
19 in 68% yield (59 mg, 0.075 mmol). 1H NMR (CDCl3), δ: 8.01
(d, J ) 2.3 Hz, 1H), 7.65 (brs, 1H), 7.62 (d, J ) 8.7 Hz, 1H),
7.44 (d, J ) 8.2 Hz, 2H), 7.30-7.10 (m, 8H), 6.72 (brs, 1H),
5.40 (s, 2H), 4.95 (s, 2H), 4.04 (t, J ) 6.6 Hz, 2H), 2.77 (t, J )
7.7 Hz, 2H), 2.69 (d, J ) 7.1 Hz, 2H), 2.09 (br t, J ) 7.0 Hz,
2H), 1.92 (m, 1H), 1.82 (m, 2H), 1.59 (m, 2H), 1.49 (m, 2H),
1.32-1.12 (m, 4H), 1.02 (t, J ) 7.2 Hz, 3H), 0.98 (d, J ) 6.6
N-Bu tyloxyca r bon yl-5-isobu tyl-3-{4-[6-(N-a cetyl-eth yl-
am in o)-4-oxo-2-pr opyl-4H-qu in azolin -3-ylm eth yl]ph en yl}-
th iop h en e-2-su lfon a m id e (15). Compound 28 (66 mg, 0.10
mmol) was used according to the general procedure and gave
after purification (isohexane:EtOAc 5:1) compound 15 in 40%
Hz, 6H), 0.85 (t, J ) 7.2 Hz, 3H), 0.81 (t, J ) 7.3 Hz, 3H); 13
C
1
yield (27 mg, 0.040 mmol). H NMR (CDCl3), δ: 8.10 (d, J )
NMR (CDCl3), δ: 172.9, 161.8, 158.0, 151.6, 150.1, 146.2, 145.9,
140.7, 137.1, 136.3, 135.0, 133.5, 130.7, 129.5, 129.4, 128.6,
128.5, 128.3, 127.4, 126.4, 126.2, 120.9, 66.8, 53.0, 46.5, 39.3,
36.9, 34.2, 30.5, 30.4, 27.5, 22.3, 22.2, 20.6, 18.7, 13.8, 13.5;
IR (compression cell), cm-1: 3500-2700(br), 3059, 2959, 1749,
1671, 1592; Anal. (C43H52N4O6S2) C, H, N.
2.3 Hz, 1H), 7.81 (d, J ) 8.4 Hz, 1H), 7.55 (m, 1H), 7.45 (d, J
) 8.3 Hz, 2H), 7.25 (d, J ) 10.0 Hz, 2H), 6.73 (s, 1H), 5.45 (s,
2H), 4.05 (t, J ) 6.6 Hz, 2H), 3.81 (q, J ) 7.1 Hz, 2H), 2.83 (t,
J ) 7.4 Hz, 2H), 2.70 (d, J ) 7.1 Hz, 2H), 1.94-1.85 (m, 6H),
1.50 (m, 1H), 1.28-1.19 (m, 2H), 1.16-1.06 (m, 6H), 0.98 (d,
J ) 6.6 Hz, 6H), 0.85 (t, J ) 7.4 Hz, 3H); 13C NMR (CDCl3), δ:
169.8, 161.1, 159.5, 151.7, 150.2, 145.8, 144.0, 141.7, 135.6,
135.2, 133.9, 130.8, 129.7, 129.3, 127.5, 126.4, 126.2, 120.5,
66.9, 46.8, 44.1, 39.3, 36.2, 30.5, 30.4, 22.9, 22.2, 21.1, 18.7,
13.8, 13.6, 13.1; IR (compression cell), cm-1:2960, 2871, 1746,
1671, 1591; Anal. (C35H44N4O6S2) C, H, N.
N-Bu tyloxyca r bon yl-5-isobu tyl-3-{4-[6-(N-ben zoyl-ben -
zyla m in o)-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl]p h e-
n yl}-th iop h en e-2-su lfon a m id e (20). Compound 33 (79 mg,
0.10 mmol) was used according to the general procedure and
gave after purification (isohexane:EtOAc 5:1) compound 20 in
1
76% yield (61 mg, 0.076 mmol). H NMR (CDCl3), δ: 8.02 (d,
N-Bu tyloxyca r bon yl-5-isobu tyl-3-{4-[6-(N-ben zoyleth -
yla m in o)-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl]p h e-
n yl}-th iop h en e-2-su lfon a m id e (16). Compound 29 (73 mg,
0.10 mmol) was used according to the general procedure and
gave after purification (isohexane:EtOAc 5:1) compound 16 in
J ) 2.6 Hz, 1H), 7.62 (brs, 1H), 7.45-7.08 (m, 16H), 6.72 (s,
1H), 5.35 (s, 2H), 5.21 (s, 2H), 4.03 (t, J ) 6.6 Hz, 2H), 2.77-
2.65 (m, 4H), 2.01-1.67 (m, 3H), 1.47 (m, 2H), 1.22 (m, 2H),
1.02-0.94 (m, 9H), 0.84 (t, J ) 7.3 Hz, 3H); 13C NMR (CDCl3),
δ: 170.6, 161.7, 157.7, 151.6, 150.0, 145.8, 141.8, 136.8, 136.1,
135.3, 134.7, 133.5, 130.8, 129.9, 129.4, 129.3, 128.8, 128.5,
128.2, 128.0, 127.5, 126.4, 124.2, 120.4, 66.8, 53.9, 46.4, 39.2,
36.6, 30.4, 30.3, 22.1, 20.5, 18.6, 13.8, 13.5; IR (compression
cell), cm-1: 2960, 2872, 1746, 1649; Anal. (C45H48N4O6S2) C,
H, N.
1
44% yield (33 mg, 0.044 mmol). H NMR (CDCl3), δ: 8.04 (d,
J ) 2.5 Hz, 1H), 7.56 (d, J ) 8.9 Hz, 1H), 7.44 (d, J ) 8.3 Hz,
2H), 7.34-7.13 (m, 8H), 6.72 (s, 1H), 5.39 (s, 2H), 4.08-4.01
(m, 4H), 2.80-2.68 (m, 4H), 1.99-1.77 (m, 3H), 1.53-1.49 (m,
2H), 1.27-11.20 (m, 5H), 1.03-0.97 (m, 9H), 0.85 (t, J ) 7.3
Hz, 3H); 13C NMR (CDCl3), δ: 170.3, 161.6, 158.5, 151.8, 150.0,
145.9, 142.1, 136.0, 135.7, 135.3, 135.1, 133.8, 130.7, 129.9,
129.6, 129.4, 128.7, 128.1, 127.0, 126.4, 124.5, 120.4, 66.9, 46.6,
45.7, 39.3, 36.5, 30.5, 30.4, 22.2, 20.9, 18.7, 13.9, 13.6, 13.1;
IR (compression cell), cm-1: 3058, 2961, 2932, 2872, 1745,
1644; Anal. (C40H46N4O6S2ׂH2O) C, H, N.
N-Bu tyloxyca r bon yl-5-isobu tyl-3-(4-{6-[N-(th iop h en e-
2-ca r bon yl)ben zyla m in o]-4-oxo-2-p r op yl-4H-qu in a zolin -
3-ylm eth yl}p h en yl)th iop h en e-2-su lfon a m id e (21). Com-
pound 34 (73 mg, 0.095 mmol) was used according to the
general procedure and gave after purification (isohexane:
EtOAc 5:1) compound 21 in 63% yield (49 mg, 0.060 mmol).
1H NMR (CDCl3), δ: 8.14 (d, J ) 2.3 Hz, 1H), 7.66 (brs, 1H),
7.61 (d, J ) 8.5 Hz, 1H), 7.44 (d, J ) 8.2 Hz, 2H), 7.36-7.16
(m, 9H), 6.90 (dd, J ) 3.8, 1.1 Hz, 1H), 6.78 (dd, J ) 5.0, 3.8
Hz, 1H), 6.73 (s, 1H), 5.39 (s, 2H), 5.13 (s, 2H), 4.04 (t, J ) 6.5
Hz, 2H), 2.79 (br t, J ) 7.4 Hz, 2H), 2.69 (d, J ) 6.9 Hz, 2H),
1.93 (m, 1H), 1.85 (m, 2H), 1.49 (m, 2H), 1.24 (m, 2H), 1.03 (t,
J ) 7.2 Hz, 3H), 0.98 (d, J ) 6.6 Hz, 6H), 0.85 (t, J ) 7.2 Hz,
3H); 13C NMR (CDCl3), δ: 162.6, 161.7, 158.4, 151.7, 150.1,
145.9, 140.9, 137.4, 136.6, 136.2, 135.8, 133.6, 132.9, 131.1,
130.7, 129.5, 129.4, 128.7, 128.5, 128.0, 127.6, 126.8, 126.5,
126.4, 120.8, 66.9, 54.7, 46.5, 39.3, 36.8, 30.5, 30.4, 22.2, 20.6,
18.7, 13.8, 13.6; IR (compression cell), cm-1: 3219, 3062, 2961,
1750, 1673, 1627, 1587; Anal. (C43H46N4O6S3) C, H, N.
Ra t Liver Mem br a n e AT1 Recep tor Bin d in g Assa y. Rat
liver membranes were prepared according to the method of
Dudley et al.17 Binding of [125I]Ang II to membranes was
conducted in a final volume of 0.5 mL containing 50 mM Tris-
HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA,
0.025% bacitracin, 0.2% BSA (bovine serum albumin), liver
homogenate corresponding to 5 mg of the original tissue
weight, [125I]Ang II (80000-85000 cpm, 0.03 nM), and variable
concentrations of test substance. Samples were incubated at
25 °C for 2 h, and binding was terminated by filtration through
Whatman GF/B glass-fiber filter sheets, which had been
presoaked overnight with 0.3% polyethylamine, using a Bran-
del cell harvester. The filters were washed with 3 × 3 mL of
Tris-HCl (pH 7.4) and transferred to tubes. The radioactivity
was measured in a gamma counter. The characteristics of the
Ang II binding AT1 receptor was determinated by using six
different concentrations (0.03-5 nmol/L) of the labeled [125I]-
AngII. Nonspecific binding was determined in the presence of
1 mM Ang II. The specific binding was determined by
subtracting the nonspecific binding from the total bound [125I]-
AngII. The apparent dissociation constant (Kd ) 1.7 ( 0.1 nM,
[L] ) 0.057 nM) were determined by Scatchard analysis of data
obtained with Ang II by using GraFit (Erithacus Software,-
UK). The binding data best fitted with a one-site fit. All deter-
minations were performed in triplicate.
N-Bu tyloxyca r bon yl-5-isobu tyl-3-(4-{6-[N-(th iop h en e-
2-ca r bon yl)eth yla m in o]-4-oxo-2-p r op yl-4H-qu in a zolin -3-
ylm eth yl}p h en yl)-th iop h en e-2-su lfon a m id e (17). Com-
pound 30 (73 mg, 0.10 mmol) was used according to the general
procedure and gave after purification (isohexane:EtOAc 5:1)
compound 17 in 93% yield (70 mg, 0.093 mmol). 1H NMR
(CDCl3), δ: 8.18 (d, J ) 2.3 Hz, 1H), 7.68 (d, J ) 8.6 Hz, 1H),
7.52 (dd, J ) 6.1, 2.5 Hz, 1H), 7.45 (d, J ) 8.1 Hz, 2H), 7.30-
7.21 (m, 3H), 6.85 (d, J ) 2.6 Hz, 1H), 6.77 (m, 1H), 6.73 (s,
1H), 5.41 (s, 2H), 4.10-3.90 (m, 4H), 2.78-2.66 (m, 4H), 1.89-
1.83 (m, 3H), 1.54-1.42 (m, 2H), 1.30-1.17 (m, 5H), 1.05-
0.94 (m, 9H), 0.85 (t, J ) 7.26 Hz, 3H); 13C NMR (CDCl3), δ:
162.2, 162.0, 157.9, 151.6, 150.2, 146.7, 145.8, 140.8, 137.9,
136.4, 135.5, 133.6, 132.5, 130.8, 130.6, 129.5, 129.4, 128.7,
126.7, 126.4, 126.3, 121.0, 66.8, 46.5, 46.2, 39.3, 37.1, 30.5, 30.4,
22.2, 20.5, 18.7, 13.9, 13.6, 12.8; IR (compression cell), cm-1
:
3074, 2961, 2872, 1747, 1672, 1591; Anal. (C38H44N4O6S3ׂ
H2O) C, H, N.
N-Bu tyloxyca r bon yl-5-isobu tyl-3-{4-[6-(N-a cetyl-ben -
zyla m in o)-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl]p h e-
n yl}-th iop h en e-2-su lfon a m id e (18). Compound 31 (75 mg,
0.11 mmol) was used according to the general procedure and
gave after purification (isohexane:EtOAc 5:1) compound 18 in
1
77% yield (63 mg, 0.085 mmol). H NMR (CDCl3), δ: 8.03 (s,
1H), 7.68-7.62 (m, 2H), 7.44 (d, J ) 8.3 Hz, 2H), 7.32-7.14
(m, 8H), 6.72 (s, 1H), 5.40 (s, 2H), 4.95 (s, 2H), 4.03 (t, J ) 6.6
Hz, 2H), 2.76 (m, 2H), 2.69 (d, J ) 7.3 Hz, 2H), 2.01-1.74 (m,
6H), 1.49 (m, 2H), 1.23 (m, 2H), 1.05-0.94 (m, 9H), 0.84 (t, J
) 7.3 Hz, 3H); 13C NMR (CDCl3), δ: 170.2, 161.6, 151.6, 150.0,
145.8, 141.0, 136.8, 136.1, 134.8, 133.6, 130.7, 129.5, 128.5,
128.1, 127.5, 126.3, 126.1, 120.8, 66.8, 52.8, 46.5, 39.2, 36.7,
30.4, 30.3, 29.6, 22.8, 22.1, 20.7, 18.7, 13.8, 13.5; IR (compres-
sion cell), cm-1: 2959, 1746, 1670, 1591; Anal. (C40H46N4O6S2)
C, H, N.
N-Bu t yloxyca r b on yl-5-isob u t yl-3-{4-[6-(N-p en t a n oyl-
ben zyla m in o)-4-oxo-2-p r op yl-4H-qu in a zolin -3-ylm eth yl]-
p h en yl}-th iop h en e-2-su lfon a m id e (19). Compound 32 (78
mg, 0.11 mmol) was used according to the general procedure
and gave after purification (isohexane:EtOAc 5:1) compound

AT2 Receptor Agonist in Vivo
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1545
P or cin e (p ig) Myom etr ia l Mem br a n e AT2 Recep tor
Ack n ow led gm en t. We gratefully acknowledge sup-
port from the Swedish Research Council (VR), the
Swedish Foundation for Strategic Research (SSF), and
Knut and Alice Wallenberg Foundation (VRmedicine
8663).
Bin d in g Assa y. Myometrial membranes were prepared from
porcine uteri according to the method by Nielsen et al.18
A
presumable interference by binding to AT1 receptors was
blocked by addition of 1 µM losartan. Binding of [125I]Ang II
to membranes was conducted in a final volume of 0.5 mL
containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM
MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2% BSA, homoge-
nate corresponding to 10 mg of the original tissue weight, [125I]-
Ang II (80000-85000 cpm, 0.03 nM), and variable concentra-
tions of test substance. Samples were incubated at 25 °C for
1.5 h and binding was terminated by filtration through
Whatman GF/B glass-fiber filter sheets, which had been
presoaked overnight with 0.3% polyethylamine, using a Bran-
del cell harvester. The filters were washed with 3 × 3 mL of
Tris-HCl (pH 7.4) and transferred to tubes. The radioactivity
was measured in a gamma counter. The characteristics of the
Ang II binding AT2 receptor were determinated by using six
different concentrations (0.03-5 nmol/L) of the labeled [125I]-
Ang II. Nonspecific binding was determined in the presence
of 1 mM Ang II. The specific binding was determined by
subtracting the nonspecific binding from the total bound [125I]-
Ang II. The apparent dissociation constant (Kd ) 0.7 ( 0.1
nM, [L] ) 0.057 nM) was determined by Scatchard analysis
of data obtained with Ang II by using GraFit (Erithacus
Software, UK). The binding data best fitted with a one-site
fit. All determinations were performed in triplicate.
Refer en ces
(1) (a) De Gasparo, M.; Catt, K. J .; Inagami, T.; Wright, J . W.;
Unger, T. International union of pharmacology. XXIII. The
angiotensin II receptors. Pharmacol. Rev. 2000, 52, 415-472.
(b) Birkenhager, W. H.; de Leeuw, P. W. Non-peptide angiotensin
type 1 receptor antagonists in the treatment of hypertension.
J . Hypertens. 1999, 17, 873-881. (c) Greenlee, W. J . Hyperten-
sion - Treatment by blockade of the renin-angiotensin system.
Pharmacochem. Lib. 1997, 28, 97-107. (d) Timmermans, P. B.
M. W. M.; Smith, R. D. Antihypertensive Agents. Burger’s
Medicinal Chemistry and Drug Discovery, 5th ed.; J ohn Wiley
& Sons: New York, 1996. (e) Wexler, R. R.; Greenlee, W. J .;
Irvin, J . D.; Goldberg, M. R.; Prendergast, K.; Smith, R. D.;
Timmermans, P. B. M. W. M. Nonpeptide angiotensin II receptor
antagonists: The next generation in antihypertensive therapy.
J . Med. Chem. 1996, 39, 625-656. (f) Ashton, W. T.; Chang, L.
L.; Flanagan, K. L.; Mantlo, N. B.; Ondeyka, D. L.; Kim, D.; de
Laszlo, S. E.; Glinka, T. W.; Rivero, R. A.; Kevin, N. J .; Chang,
R. S. L.; Siegl, P. K. S.; Kivlighn, S. D.; Greenlee, W. J . AT1/
AT2-balanced angiotensin II antagonists. Euro. J . Med. Chem.
1995, 30, 255-266. (g) Ashton, W. T. Nonpeptide angiotensin
II receptor antagonists. Exp. Opin. Invest. Drugs 1994, 3, 1105-
1142. (h) Duncia, J . V.; Carini, D. J .; Chiu, A. T.; J ohnson, A.
L.; Price, W. A.; Wong, P. C.; Wexler, R. R.; Timmermans, P. B.
M. W. M. The discovery of DuP 753, a potent, orally active
nonpeptide angiotensin II receptor antagonist. Med. Res. Rev.
1992, 12, 149-191.
In Vivo Settin g. Gen er a l. The in vivo experiments were
approved by the Animal Ethics Committee of Gothenburg
University and performed on nonfasted Male Sprague-Dawley
rats (Mo¨llegård Breeding Center Ltd., Ejby, Denmark). For
induction of anesthesia, pentobarbital, was injected intrap-
eritoneally (60 mg kg-1 bw). General anesthesia was main-
tained with R-chloralose administered intravenously as a bolus
(50 mg kg-1 bw) followed by continuous infusion (25 mg kg-1
h-1). A catheter was inserted into the trachea to ensure free
airways. A femoral artery and one or two veins were cath-
eterized for subsequent blood pressure measurements and
drug infusions, respectively. The body temperature was main-
tained at 38 °C with a heating pad and lamp, both controlled
thermostatically. Blood pressure was measured by a Statham
P23Dc transducer (Statham, Hato Rey, Puerto Rico) connected
to a PE-50 catheter in the right femoral artery. Pressure data
were integrated by a microcomputer to mean arterial pressure
(MAP) over 5 min. To avoid acidosis and dehydration due to
the surgical trauma and the long period of general anesthesia,
an 1.7% glucose solution containing 0.03 M NaHCO3, made
isotonic with saline, was given intravenously throughout the
experiments (1 mL h-1). Vehicle used for losartan: 150 mM
NaCl(aq), compound 1: 1% EtOH in 150 mM NaCl(aq) and
(2) (a) Perlman, S.; Schambye, H. T.; Rivero, R. A.; Greenlee, W.
J .; Hjorth, S. A.; Schwartz, T. W. Non-peptide angiotensin
agonist. Functional and molecular interaction with the AT1
receptor. J . Biol. Chem. 1995, 270, 1493-1496. (b) Kivlighn, S.
D.; Huckle, W. R.; Zingaro, G. J .; Rivero, R. A.; Lotti, V. J .;
Chang, R. S. L.; Schorn, T. W.; Kevin, N.; J ohnson, R. G., J r.;
Greenlee, W. J .; Siegl, P. K. S. Discovery of L-162,313:
a
nonpeptide that mimics the biological actions of angiotensin II.
Am. J . Physiol. 1995, 268, R820-R823.
(3) Perlman, S.; Costa-Neto, C. M.; Miyakawa, A. A.; Schambye, H.
T.; Hjorth, S. A.; Paiva, A. C. M.; Rivero, R. A.; Greenlee, W. J .;
Schwartz, T. W. Dual agonistic and antagonistic property of
nonpeptide angiotensin AT1 ligands: susceptibility to receptor
mutations. Mol. Pharmacol. 1997, 51, 301-311.
(4) Huckle, W. R.; Kivlighn, S. D.; Zingaro, G. J .; Kevin, N. J .;
Rivero, R. A.; Chang, R. S. L.; Greenlee, W. J .; Siegl, P. K. S.;
J ohnson, R. G. Angiotensin II receptor-mediated activation of
phosphoinositide hydrolysis and elevation of mean arterial
pressure by a nonpeptide, L-163, 491. Can. J . Physiol. Pharma-
col. 1994, 72, 543.
(5) Gallinat, S.; Busche, S.; Raizada, M. K.; Sumners, C. The
angiotensin II type 2 receptor: an enigma with multiple varia-
tions. Am. J . Physiol. 2000, 278, E357-E374.
(6) de Gasparo, M.; Siragy, H. M. The AT2 receptor: fact, fancy and
fantasy. Regul. Peptides 1999, 81, 11-24.
(7) J ohansson, B.; Holm, M.; Ewert, S.; Casselbrant, A.; Pettersson,
A.; Fandriks, L. Angiotensin II type 2 receptor-mediated duode-
nal mucosal alkaline secretion in the rat. Am. J . Physiol. 2001,
280, G1254-G1260.
(8) Blankley, C. J .; Hodges, J . C.; Klutchko, S. R.; Himmelsbach,
R. J .; Chucholowski, A.; Connolly, C. J .; Neergaard, S. J .; Van
Nieuwenhze, M. S.; Sebastian, A.; Quin, J ., III. Synthesis and
structure-activity relationships of a novel series of nonpeptide
angiotensin II receptor binding inhibitors specific for the AT2
subtype. J . Med. Chem. 1991, 34, 3248-3260.
(9) Carini, D. J .; Duncia, J . V.; Aldrich, P. E.; Chiu, A. T.; J ohnson,
A. L.; Pierce, M. E.; Price, W. A.; Santella, J . B., 3rd; Wells, G.
J .; Wexler, R. R.; Wong, P. C.; Yoo, W.-E.; Timmermans, P. B.
M. W. M. Nonpeptide angiotensin II receptor antagonists: the
discovery of a series of N-(biphenylylmethyl)imidazoles as potent,
orally active antihypertensives. J . Med. Chem. 1991, 34, 2525-
2547.
compound 11: 1% DMSO in 150 mM NaCl(aq)
.
Secr etion . Duodenal mucosal alkaline (HCO3-) secretion
was measured by a pH-stat titration technique. This technique
has been described previously,14 but a brief summary will be
given here. After midline laparotomy, a segment of the
duodenum, with its proximal end approximately 1 cm distal
to the pylorus, with intact vascular supply was isolated
between two glass tubes connected to a reservoir containing
isotonic saline maintained at 38 °C by a water-jacket. Saline
was recirculated through the segment by means of a gas-lift
(pure air). The common bile duct was catheterized approxi-
mately 5 mm proximal to the papilla of water, to avoid
contamination of the segment by bile and pancreatic juice.
Alkaline secretion to the perfusate was continuously titrated
to pH 7.4 with 0.02 M HCl controlled by a pH-stat device.
(10) (a) de Laszlo, S. E.; Quagliato, C. S.; Greenlee, W. J .; Patchett,
A. A.; Chang, R. S. L.; Lotti, V. J .; Chen, T. B.; Scheck, S. A.;
Faust, K. A.; Kivlighn, S. D.; Schorn, T. S.; Zingaro, G. J .; Siegl,
P. K. S. A potent, orally active, balanced affinity angiotensin II
AT1 antagonist and AT2 binding inhibitor. J . Med. Chem. 1993,
36, 3207-3210. (b) Glinka, T. W.; de Laszlo, S. E.; Tran, J .;
Chang, R. S.; Chen, T. B.; Lotti, V. J .; Greenlee, W. J . L-161,-
638: a potent AT2 selective quinazolinone angiotensin II binding
inhibitor. Bioorg. Med. Chem. Lett. 1994, 4, 1479-1484. (c)
Glinka, T. W.; de Laszlo, S. E.; Siegl, P. K. S.; Chang, R. S.;
Kivlighn, S. D.; Schorn, T. S.; Faust, K. A.; Chen, T. B.; Zingaro,
Sta tistics. Changes in alkaline secretion and mean arterial
pressure were analyzed by ANOVA (Bonferroni post hoc). Data
obtained during the last 15 min before administration of drug
were regarded as basal conditions. Net change was defined
as the difference between basal conditions and data obtained
between 15 min to 30 min following onset of each drug
administration. Comparisons between groups were made by
one-way ANOVA and a t-test. Values given in the figures are
means ( SEM. A p-value e 0.05 was considered significant.

1546 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wan et al.
G. J .; et al. A new class of balanced AT1/AT2 angiotensin II
antagonists: quinazolinone AII antagonists with acylsulfona-
mide and sulfonylcarbamate acidic functionalities. Bioorg. Med.
Chem. Lett. 1994, 4, 81-86. (d) Chakravarty, P. K.; Strelitz, R.
A.; Chen, T. B.; Chang, R. S. L.; Lotti, V. J .; Zingaro, G. J .;
Schorn, T. W.; Kivlighn, S. D.; Siegl, P. K. S.; Patchett, A. A.;
(13) (a) Dudley, D. T.; Panek, R. L.; Major, T. C.; Lu, G. H.; Bruns,
R. F.; Klinkefus, B. A.; Hodges, J . C.; Weishaar, R. E. Subclasses
of angiotensin II binding sites and their functional significance.
Mol. Pharmacol. 1990, 38, 370-377. (b) Nielsen, A. H.; Schauser,
K.; Winther, H.; Dantzer, V.; Poulsen, K. Angiotensin II recep-
tors and renin in the porcine uterus: myometrial AT2 and
endometrial AT1 receptors are down-regulated during gestation.
Clin. Exp. Pharmacol. Physiol. 1997, 24, 309-314.
(14) Flemstro¨m, G.; Garner, A.; Nylander, O.; Hurst, B. C.; Heylings,
J . R. Surface epithelial HCO3(-) transport by mammalian
duodenum in vivo. Am. J . Physiol. Gastrointest. Liver Physiol.
1982, 243, G348-358.
(15) Mantlo, N. B.; Kim, D.; Ondeyka, D. L.; Chang, R. S. L.; Kivlighn,
S. D.; Sieg, P. K. S.; Greenlee, W. J . Imidazo[4,5-b]pyridine-based
AT1/AT2 angiotensin II receptor antagonists. Bioorg. Med.
Chem. Lett. 1994, 4, 17-22.
(16) J ohansson, B.; Holm, M.; Chen, L. H.; Pettersson, A.; J onson,
C.; Fandriks, L. ANG II prolongs splanchnic nerve-mediated
inhibition of duodenal mucosal alkaline secretion in the rat. Am.
J . Physiol.-Regul. Integr. Comput. Physiol. 1997, 273, R942-
R946.
(17) Dudley, D. T.; Panek, R. L.; Major, T. C.; Lu, G. H.; Bruns, R.
F.; Klinkefus, B. A.; Hodges, J . C.; Weishaar, R. E. Subclasses
of angiotensin-II binding-sites and their functional-significance.
Mol. Pharmacol. 1990, 38, 370-377.
(18) Nielsen, A. H.; Schauser, K.; Winther, H.; Dantzer, V.; Poulsen,
K. Angiotensin II receptors and renin in the porcine uterus:
Myometrial AT(2) and endometrial AT(1) receptors are down-
regulated during gestation. Clin. Exp. Pharmacol. Physiol. 1997,
24, 309-314.
Greenlee, W. J . Quinazolinone biphenyl acylsulfonamides:
a
potent new class of angiotensin-II receptor antagonists. Bioorg.
Med. Chem. Lett. 1994, 4, 75-80. (e) de Laszlo, S. E.; Chang, R.
S.; Chen, T.-B.; Faust, K. A.; Greenlee, W. J .; Kivlighn, S. D.;
Lotti, V. J .; O’Malley, S. S.; Schorn, T. W.; et al. The SAR of
6-(N-alkyl-N-acyl)-2-propyl-3-[(2′-(tetrazol-5-yl)biphen-4-yl)meth-
yl]quinazolinones as balanced affinity antagonists of the human
AT1 and AT2 receptors. Bioorg. Med. Chem. Lett. 1995, 5, 1359-
1364. (f) de Laszlo, S. E.; Glinka, T. W.; Greenlee, W. J .; Ball,
R.; Nachbar, R. B.; Prendergast, K. The design, binding affinity
prediction and synthesis of macrocyclic angiotensin II AT1 and
AT2 receptor antagonists. Bioorg. Med. Chem. Lett. 1996, 6,
923-928. (g) Levin, J . I.; Chan, P. S.; Porter, R. S. 2,3,6-
Substituted quinazolinones as angiotensin II receptor antago-
nists. Drugs Future 1995, 20, 55-65.
(11) (a) Kevin, N. J .; Rivero, R. A.; Greenlee, W. J .; Chang, R. S. L.;
Chen, T. B. Substituted phenylthiophene benzoylsulfonamides
with potent binding affinity to angiotensin II AT1 and AT2
receptors. Bioorg. Med. Chem. Lett. 1994, 4, 189-194. (b)
Kivlighn, S.; Lotti, V. J .; Rivero, R. A.; Siegl, P. K. S.; Zingaro,
G. J . Preparation of thiophene-substituted imidazo[4,5-b]pyri-
dine angiotensin II receptor agonists. British UK Pat. Appl. GB
2281298 (Merck and Co., Inc., Rahway, NJ ) 1995.
(12) Senanayake, C. H.; Fredenburgh, L. E.; Reamer, R. A.; Liu, J .;
Roberts, F. E.; Humphrey, G.; Thompson, A. A.; Larsen, R. D.;
Verhoeven, T. R.; Reider, P. J .; Sinkai, I. New approach to the
imidazolutidine moiety of MK-996. Heterocycles 1996, 42, 821-
830.
J M031031I